Five related aspects of neuronal development and synapse formation will be examined in associated cell cultures prepared with embryonic chick ciliary ganglion (CG) neurons. These include (1) the normal determinants of neuronal survival, (2) the specification of neurotransmitter synthesis, (3) the regulation of the neuronal alpha-bungarotoxin binding component and its relation to the neuronal acetylcholine (ACh) receptor, (4) the formation and maintenance of nerve-nerve and nerve-muscle synapses, and (5) the neuronal regulation of myotube ACh receptors. When grown alone in cell culture with muscle- or heart-conditioned medium, DCG neurons exhibit quantitative long-term survival, including those neurons that were destined to die in ovo as part of normal ganglionic development. The factor(s) present in conditioned medium that are responsible for the survival will be purified and their mode of action characterized. Neurotransmitter synthesis will be examined to identify the events that commit CG neurons to a cholinergic fate. An early developmental stage will be sought in which the neurons retain the capacity to express other differentiated properties characteristic of neuronal crest derivatives such as catecholamine biosynthesis. The binding of alpha-bungarotoxin to the neurons will be studied to distinguish the different classes of binding and to determine their relationship to the neuronal ACh receptor. Synapse formation in CG neuron-myotube cultures will be examined (1) to assess the stability of individual points of nerve-muscle synaptic contact, (b) to determine whether synaptic activity can prevent the formation of additional synapses on an innervated myotube, and (c) to determine whether CG neurons can innervate each other in cell culture. The neuronal regulation of myotube ACh receptors will be examined to identify the mechanisms underlying the induction of receptors and to determine whether the neurons also promote a re-distribution of the receptors in the myotube membrane. Fluorescent- and radiolabeled-toxins together with iontophoretic application of ACh will be used for the receptor analysis.